Method for determining release of a peptide from a sustained release polylactide formulation

ABSTRACT

A method for determining the release of a peptide from a sustained release polylactide formulation which can be carried out in a shorter time than a real-time method by accelerating the release of the peptide from the formulation. It can be used to discriminate between batches of formulations for quality control purposes and to discriminate between formulations, for example, having different drug loadings.

[0001] This application is a national stage filing under 35 U.S.C. 371of International Application No. PCT/GB01/04439, filed Oct. 5, 2001,which claims priority from United Kingdom Application No. 0024884.9,filed Oct. 11, 2000, the specifications of each of which areincorporated by reference herein. International Application No.PCT/GB01/04439 was published under PCT Article 21(2) in English.

[0002] The present invention relates to an accelerated method fordetermining the release of a peptide from a sustained releasepolylactide formulation.

[0003] The therapeutic effectiveness of pharmaceutically active drugssuch as peptides may depend on their continued release over a prolongedperiod of time. It is recognised that a single administration of apharmaceutical composition, comprising such a drug, which results inrelease of the drug over an extended period of time, for example onemonth or more, has significant clinical advantages, for example avoidingthe need for repeated administrations. Such extended or sustainedrelease can be achieved by administering the drug in the form of abiodegradable polymer matrix which releases the drug over a prolongedperiod. Suitable biodegradable polymers are polylactides. The term‘polylactide’ is used in a generic sense to include polymers of lacticacid alone, copolymers of lactic and glycolic acid, mixtures of suchpolymers, mixtures of such copolymers and mixtures of such polymers andcopolymers, the lactic acid being either in racemic or optically activeform.

[0004] The polylactide polymer may be prepared using known methods.Suitable techniques include, but are not limited to, condensationpolymerisation of the appropriate hydroxy acids, i.e. lactic andglycolic acids, and ring opening polymerisation of the appropriatelactide and glycolide dimers.

[0005] Examples of peptides which are beneficially administered in asustained release formulation include LHRH analogues which can be usedin the treatment of a number of diseases and clinical disorders. Anexample of one such disease is prostate cancer, a sex hormone dependentcancer. This can be treated by chemical means by interfering with theproduction of luteinising hormone (LH) which regulates the synthesis ofmale hormones. Luteinising hormone releasing hormone (LHRH) is a naturalhormone, produced by the hypothalamus, that interacts with theluteinising hormone releasing hormone receptor in the pituitary tostimulate the production of LH. Administration of an LHRH analogue willdisturb the production of the LH. Such LHRH analogues have similarlybeen used to treat, for example, estrogen-dependent cancers such asbreast cancer. Examples of such LHRH analogues include, for example,leuprorelin (U.S. Pat. No. 4,005,063), goserelin (U.S. Pat. No.4,100,274), triptorelin (U.S. Pat. No. 4,010,125) and buserelin (U.S.Pat. No. 4,024,248).

[0006] Examples of commercially available LHRH analogues include‘Zoladex’, which contains goserelin as the active ingredient, and‘Prostap 3’, which contain leuprorelin as the active ingredient.

[0007] Another class of peptide derivatives which are beneficiallyadministered in a sustained release formulation include Sandostatin, acommercially available form of octreotide and Somatuline, a commerciallyavailable form of lanreotide, both of which are somatostatin analogues.

[0008] Another class of peptide derivatives which possesspharmacologically beneficial properties which can be used in treatingautoimmune diseases or medical conditions, such as rheumatoid arthritisand other MHC class II dependent T-cell mediated diseases and which mayusefully be administered from a sustained release composition are thosecompounds described in WO 97/31023, published 28 Aug. 1997, incorporatedherein by reference.

[0009] Whilst real-time in-vivo release studies are essential toevaluate sustained release compositions, a discriminatory in-vitromethod is essential to evaluate differences between batches for qualitycontrol purposes. Whilst such in-vitro methods are typically real timetests typically performed at 37° C., it is advantageous if anaccelerated method can be applied to confirm the quality of clinicaltrials materials or marketed products. The results of the test would becompared with specification limits, typically at three or moretime-points, chosen to ensure that all batches released will beeffective in terms of onset, maintenance and duration of action. Such anaccelerated in-vitro method may also be used in product development, toevaluate the effect of modifications to composition or manufacturingprocess, thus shortening development time-lines and minimising the needfor in-vivo studies. An important aspect is to ensure that completerelease of the peptide is achieved during the duration of theaccelerated method. By ‘complete release’ is meant that at least 90%,preferably 95% or more, of the peptide is released during the durationof the test. Another important feature is to ensure that the short-termor accelerated release correlates with real-time release, i.e. similarrelease profiles of drug from the formulation are achieved, in orderthat the short-term release can be used as predictive of real-timerelease. By ‘similar-release profiles’ is meant the release profile fromthe real-time and accelerated tests are essentially the same shape. Forexample, if the release profile from the real-time method is consistentwith, for example, two-phase release the release profile from theaccelerated method should also show two phases of release. The use ofsuch an accelerated method may mean in the case of a three monthsustained release formulation, that individual formulations could becompared after a matter of a few days rather than waiting for areal-time analysis taking around 90 days.

[0010] Such accelerated methods have previously been described. Forexample, McDaniel, Proceed. Int'l. Symp. Control. Rel. Bioact. Mater.,25, 1998, p443, have disclosed an accelerated in-vitro release methodfor microsphere formulations containing a water-insoluble analogue ofthe avermectin family of antibiotics in a polylactide glycolide polymermatrix. This method employs one of a range of organic amines ascatalysts to accelerate degradation of the polymer. The use of hightemperatures, approaching the glass transition temperature (Tg) of thepolymer, to further accelerate degradation of the polymer was rejected.

[0011] Shameem et al in AAPS Pharmsci 1999, 1(3), article 7 describes anaccelerated method to evaluate peptides from polylactide glycolidemicrosphere formulations. In this method it was found necessary to usetemperatures which were above the Tg of the polymer to ensure completerelease of the peptide. The dissolution medium was optimised at pH 4 andpolyvinylalcohol was present as a surfactant to prevent the microspheresfrom sticking together. Complete release of the peptide was typicallyachieved in the accelerated test within 30 to 40 hours whereas real-timerelease was 28 days.

[0012] The methods disclosed in these references lead in two opposeddirections since one teaches the use of the temperatures above the Tg ofthe polymer whereas the other teaches temperatures below the Tg of thepolymer to ensure accelerated degradation of the polymer. Furthermore,neither reference identifies an accelerated test for a 1 monthformulation which gives complete release of the peptide and can becarried out in less than 30 hours. Clearly, it is advantageous if theaccelerated method can be carried out in 15 hours or less and preferably12 hours or less since it enables the test to be complete withinslightly extended working day.

[0013] Thus, according to the invention there is provided a method fordetermining the release of a peptide from a sustained releasepolylactide formulation, which can be carried out in a shorter time thana real-time method by accelerating the release of the peptide from theformulation, the method comprising

[0014] i) adding the formulation to a fixed amount of dissolutionmedium, the dissolution medium having an osmolarity in the range 100 to550 mOsmol/L and pH in the range 7 to 12, and, optionally, comprisingone or more further components selected from catalysts and surfactants;

[0015] ii) maintaining the temperature of the dissolution medium withinthe range 0 to 50° C. above the Tg of the polylactide polymer; and

[0016] iii) determining the amount of peptide in the sample,

[0017] the osmolarity, pH and temperature being chosen such that theaccelerated release profile is similar to the real-time release profile.

[0018] The method can be used to discriminate between batches offormulations for quality control purposes and or to discriminate betweenformulations, for example, those having different drug loadings or beingformed from different polymers.

[0019] Preferably, the dissolution medium will be sampled at regulartime intervals, such as for example, at hourly time intervals.

[0020] The sustained release formulation may be in the form of a solidcomposition which may be administered to patients by subdermalimplantation or by injection or in the form of a suspension forinjection comprising microparticles or microcapsules. Such compositionsare described in, amongst other references, European Patents 58 481 and52 510, which are incorporated herein by reference. When the formulationis in the form of a suspension for injection, the dissolution mediumwill preferably additionally include a surfactant which assists inpreventing agglomeration of the microparticles or microcapsules whichmay occur, particularly at higher temperatures. The use of a surfactantcan, however, also be advantageously added to the dissolution mediumwhen solid compositions are being tested. Suitable surfactants includepolyvinyl alcohol, sodium dodecyl sulphate and Polysorbate 80(polyoxyethylene sorbitan monooleate). The surfactant, if present, ispreferably included at a level up to 0.5 and, in particular, from 0.05to 0.5% by weight. The accelerated method is particularly suitable formonolithic implants also known as monolithic depot formulations of thetype described in EP 58 481.

[0021] The accelerated method is particularly suitable for formulationsin which the polylactide is a lactide glycolide copolymer having alactide to glycolide ratio in the range 1:1 to 19:1 and an averagemolecular weight in the range between 10 and 150 kDa and where the drugloading is between 3 and 30% by weight, preferably between 8 and 30% byweight.

[0022] In one embodiment of the method according to the invention, thedissolution medium is sampled whilst the volume of the dissolutionmedium is maintained at constant volume.

[0023] Thus, according to a further aspect of the invention there isprovided a method for determining the release of a peptide from asustained release polylactide formulation, which can be carried out in ashorter time than a real-time method by accelerating the release of thepeptide from the formulation, the method comprising

[0024] i) adding the formulation to a fixed amount of dissolutionmedium, the dissolution medium having an osmolarity in the range 200 to500 mOsmol/L, pH in the range 7 to 12 and, optionally, comprising acatalyst such as an organic amine;

[0025] ii) maintaining the temperature of the dissolution medium withinthe range 0 to 50° C. above the Tg of the polylactide polymer;

[0026] iii) sampling the dissolution medium whilst maintaining thevolume of the dissolution medium constant; and

[0027] iv) determining the amount of peptide in the sample,

[0028] the osmolarity, pH and temperature being chosen such that theaccelerated release profile is similar to the real-time release profile.

[0029] The osmolarity, pH and temperature of the dissolution medium willdepend on the nature of the formulation being tested. For any particularformulation, initially it is necessary to establish an in-vivo releaseprofile ideally in humans but often using an animal model (rat, dog orother species as considered appropriate for the indication. Such aprofile will typically be based on pharmacokinetic data derived frommonitoring the concentration of the active ingredient in blood over aperiod exceeding the anticipated duration of release from the product).Alternatively a real time in-vitro dissolution test can be used toobtain a real-time release profile. The conditions for an acceleratedmethod can then be established by varying the osmolarity, pH andtemperature of the dissolution medium within the ranges hereinbeforedefined until the release profile from the accelerated method is similarto the real-time release profile and the duration of the method isacceptable. Formulations and or manufacturing conditions may be modifiedand the effect of such modifications assessed in-vitro using theaccelerated test, thus minimising the need for real time in-vivostudies. As stated above, it is advantageous if the conditions can bechosen such that the duration of the test is complete within a workingday, particularly if the dissolution medium is sampled manually.Furthermore, if the method is being used for rank correlation ofclinical batches it is important that this can be completed in as shorta time as possible since ultimately this may reduce the delay betweenbatch production and release of product into the market.

[0030] Preferably, release of the peptide from a 1 month sustainedrelease formulation will be accelerated to be complete in less than 12hours. For three and six month sustained release formulations theaccelerated method will preferably aim to give complete release of thepeptide within less than 25 hours and 50 hours respectively.

[0031] Preferably the peptide is an LHRH analogue, octreotide,lanreotide or a MHC class II antagonist. Most preferably the peptide isgoserelin or an MHC class II antagonist.

[0032] As stated above, for the accelerated method to be useful it isimportant that the release profile using the accelerated method issimilar to that obtained using the real-time method. Whilst we havefound that increasing the temperature at which the method is carried outto temperatures greater than 50° C. above the Tg of the polymer may leadto a faster release of peptide, such high temperatures may result in aloss of the integrity and physical form of the formulation which islikely to affect the release profile. Preferably the temperature of thedissolution medium will be in the range 5 to 30° C., more preferably 10to 30° C., most preferably 10 to 25° C. above the Tg of the polymer.

[0033] Suitable catalysts are organic amines which are preferablyselected from piperidine, n-methylpiperidine and piperazine. Piperidineis particularly preferred. The organic amine, if present, is preferablypresent in an amount up to 1% by weight, suitably from 0.1 to 1% byweight.

[0034] The dissolution medium comprises a buffer system suitable tomaintain the pH at the desired value. Preferably the pH of thedissolution medium will be in the range greater than pH 7, such as pH7.4, to pH 12 and more preferably pH 8 to pH 11. An ionic strengthadjuster, typically sodium chloride, may also be present to ensure theosmolarity of the dissolution medium is maintained at a suitable level.Osmolarity of the dissolution medium may be determined by the methoddescribed for ideal osmolar concentration in the US Pharmacopoeia.Preferably the osmolarity will be in the range 280 to 500 mOsmol/L, mostpreferably 400 to 500 mOsmo/L.

[0035] In one embodiment, the method can be operated manually by addingthe formulation to be tested into a vessel containing the dissolutionmedium. The temperature of the vessel and its contents can be maintainedby placing it in, for example, a water bath. At regular intervals, forexample, at hourly intervals, an aliquot of dissolution medium isremoved from the vessel and replaced with fresh dissolution medium sothat the total volume remains constant.

[0036] The absorbance of the sample can be measured using a UVspectrophotometer.

[0037] The % dissolution at the particular time interval is calculatedas follows:—

[0038] The absorbance of sample solutions n₁, n₂, n₃, . . . n_(r) . . .n_(x) is A₁, A₂, A₃ . . . A_(r) . . . A_(x) where n_(r) is before n_(x)and A_(r) is absorbance at time point n_(r) which is earlier than n_(x).${\% \quad {dissolution}} = \frac{\left\lbrack {\left( {A_{x} \times F \times V_{M}} \right) + {V_{S}F{\sum\limits_{r = 1}^{r = {x - 1}}\quad {Ar}}}} \right\rbrack \times 100}{W_{p}}$

[0039] where,

[0040] % dissolution is the cumulative percentage of nominal drugcontent release by time point x

[0041] F=Absorbance of 1 mg of the active in 1 ml of dissolution medium

[0042] W_(p)=Nominal content, in mg, of the drug in the formulationbeing tested

[0043] Ax=Absorbance at time point n_(x)

[0044] Ar=Absorbance at time point n_(r) which is earlier than n_(x)

[0045] V_(M)=total volume of the dissolution medium

[0046] V_(S)=sample volume

[0047] In an alternative embodiment, the manual operation can bereplaced by the use of commercially available equipment which relies onthe use of a flow cell such as USP Apparatus IV, commercially availablefrom Sotax or Erweka, and as described in United States Pharmacopeia23<724>Drug Release.

IN THE FIGURES

[0048]FIG. 1 is a plot of the real-time release profile of a formulationshowing % dissolution of drug in the dissolution medium release versustime.

[0049]FIG. 2 is a plot of the accelerated release profile of aformulation showing % dissolution of drug in the dissolution mediumrelease versus time.

[0050]FIG. 3 is a plot comparing the release profiles for Zoladex 3.6 mgdepot obtained for a real-time in-vitro dissolution test against thatfor an accelerated release test.

[0051]FIG. 4 is a plot showing accelerated release profiles for a numberof different monolithic depot formulations.

[0052]FIG. 5 is a plot showing the relationship between the duration ofaction and time to plateau for the different monolithic depotformulations shown in FIG. 4.

[0053]FIG. 6 is a plot comparing the accelerated release profile for a 3month microsphere formulation with Zoladex 10.8 mg.

The invention will now be illustrated by reference to the followingnon-limiting examples. EXAMPLES Example 1

[0054] A commercially available sample of ‘Zoladex’ 3.6 mg (whichcontains goserelin acetate in a polylactide glycolide copolymer) wasused in this example. The real-time dissolution of this sample wasmeasured using an in vitro real-time test.

[0055] The accelerated method conditions for a sample of ‘Zoladex’ 3.6mg were determined. The buffer used to maintain the pH at the requiredlevel of 8.0 comprised citric acid, boric acid, phosphoric acid andsodium hydroxide. Piperidine (4 ml) was added to catalyse release of thepeptide from the polymer matrix.

[0056] The osmolarity was determined to be 500 mOsMol/1 and thetemperature was 65° C. which is approximately 23° C. above the Tg of thepolymer.

[0057]FIG. 2 shows that complete release occurs in 10 hours.

[0058] ‘Zoladex’ is a Trade Mark of the AstraZeneca group of companies.

[0059] Zoladex 3.6 mg is a one month duration of action monolithic depotformulation. Zoladex 10.8 mg is a three month duration of actionmonolithic depot formulation.

Example 2

[0060] A further experiment was carried out with another sample of‘Zoladex’ 3.6 mg. In this example, the pH was maintained at pH 7.4 usinga buffer comprising potassium dihydrophosphate, disodium orthophosphateand sodium chloride. Sodium dodecyl sulphate were also added. Theosmolarity was determined to be 500 mOsMol/1 and the temperature was 65°C. which is approximately 23° C. above the Tg of the polymer. Theresults comparing the release profile for Zoladex 3.6 mg depot under theconditions of the real-time in vitro dissolution test with that obtainedfor the accelerated release test are as shown in FIG. 3.

Example 3

[0061] The relationship between duration of action and rate of releasein the accelerated release test has been investigated for the marketedproducts Zoladex 3.6 mg and Zoladex 10.8 mg. An experimental Zoladexformulation of 6-months duration made as described below and anexperimental monolithic depot formulation of the experimental MHC ClassII antagonist as described in WO 97/31023 have also been studied.

[0062] The 6 month goserelin containing formulation was prepared by meltextrusion of a freeze-dried mixture of drug and polymer. Batch sizes of5 grams were prepared by dissolving 1.16 grams of goserelin acetate and3.84 grams of a 46.2 kDa poly(dl lactide) in glacial acetic acid anddrop freezing into liquid nitrogen. The resulting frozen spheroids werefreeze dried for approximately 24 hours using a freeze dryer (Edwards),to remove glacial acetic acid from the samples. The resulting solid wasthen put through a secondary drying step as follows. Temperature wasramped from 20° C. from 45° C. over an hour followed by dwell time of 5hours. Temperature was then ramped again over 1 hour from 45° C. to 60°C. and then held at 60° C. for a period of 5 hours. Samples wereretained in a vacuum dessicator prior to extrusion. The drug/polymerblend prepared in this manner was extruded using a one gram capacity,stainless steel extruder consisting of a barrel/nozzle, base and diewith a nozzle diameter of 2.3 mm. The extruder was heated using a pipeclamp, the temperature of which was controlled through a feedbackcircuit containing a thermocouple, located in thermal contact with theextruder barrel, and a custom built controller unit. Pressure wasapplied to the extruder die via a standard hydraulic KBr Press (Specac).Extrusion pressure was controlled manually. The temperature of theextruder was initially raised to 95° C. over a period of 60 minutes, andthen held at this temperature for a further 120 minutes. Extrusion ofthe drug/polymer melt was then performed by applying a pressure ofapproximately 1 ton to the extruder die. The resulting extrudate was cutinto the required lengths for dissolution testing.

[0063] The conditions for this experiment of this example were chosen sothat release from the Zoladex 3.6 mg depot was complete in approximately3 hours.

[0064] The buffer used to maintain the pH comprised citric acid, boricacid, phosphoric acid and sodium hydroxide. The resulting acceleratedrelease profiles are presented in FIG. 4.

[0065] It is apparent that under these conditions release occurs morerapidly for the products of shorter duration. The relationship betweenduration of action and time to plateau (complete release) in theaccelerated release test is shown in FIG. 5.

[0066] The ability to rapidly assess duration of action may be ofbenefit in formulation optimisation studies in allowing the preliminaryscreening of trial formulations prior to proof of concept in animal orhuman models.

Example 4

[0067] In this example an accelerated release profile was measured forZoladex 10.8 mg and compared with an accelerated release profile forProstap 3, a 3 month duration of action commercially availablemicroparticulate formulation containing leuprorelin as the activecomponent. The experiments were carried out at pH 12. The buffer used tomaintain the pH at the required level of 8.0 comprised citric acid,boric acid, phosphoric acid and sodium hydroxide. The osmolarity wasdetermined to be 200 mOsMol/1 and the temperature was 65° C.

[0068] The results are shown in FIG. 6 and confirm that the acceleratedrelease method is applicable to microparticulate as well as monolithicdepot formulations.

1. A method for determining the release of a peptide from a sustainedrelease polylactide formulation, which can be carried out in a shortertime than a real-time method by accelerating the release of the peptidefrom the formulation, the method comprising i) adding the formulation toa fixed amount of dissolution medium, the dissolution medium having anosmolarity in the range 100 to 550 mOsmol/L and pH in the range 7 to 12;ii) maintaining the temperature of the dissolution medium within therange 0 to 50° C. above the Tg of the polylactide polymer; and iii)determining the amount of peptide in the sample, the osmolarity, pH andtemperature being chosen such that the accelerated release profile issimilar to the real-time release profile.
 2. A method according to claim1 wherein the dissolution medium further comprises one or more additivesselected from catalysts and surfactants.
 3. A method according to claim1 wherein the osmolarity of the dissolution medium is in the range 280to 500 mOsmol/L.
 4. A method according to claim 2 wherein the osmolarityof the dissolution medium is in the range 280 to 500 mOsmol/L.
 5. Amethod according to claim 1 wherein the pH of the dissolution medium isin the range 7.4 to
 12. 6. A method according to claim 1 wherein thetemperature of the dissolution medium is maintained within the range 10to 30° C. above the Tg of the polylactide polymer.
 7. A method accordingto claim 1 wherein the peptide is selected from LHRH analogues,somatostatin analogues and a peptidic MHC class II antagonist.
 8. Amethod according to claim 1 wherein the peptide is an LHRH analogue. 9.A method according to claim 1 wherein the peptide is goserelin.
 10. Amethod according to claim 1 wherein the method comprises sampling thedissolution medium whilst maintaining the volume thereof constant.
 11. Amethod according to claim 1 wherein the sustained release formulation isin the form of a monolithic depot formulation.